Getting It Right Involves More Than Simply Multiplying Vessel Size and Equipment

Might of the Unliving Dead (Cells)

Cell-free protein synthesis has been one of the more interesting options for protein production, but the method suffered from a lack of scalability. That is rapidly changing.

Research biologists routinely use cell-free preps to generate small quantities of protein for experimentation and characterization. Sutro Biopharma, which recently licensed cell-free intellectual property from James Swartz, Ph.D., of the chemical engineering department at Stanford, has engineered the technology to permit cell-free scale-up to thousands of liters and beyond.

The process essentially hijacks the protein-producing mechanism of nonliving E. coli organisms. Organisms are lysed and minimally treated to preserve transcription and translation capability, then frozen for storage and shipping.

Sutro employs glucose, glutamate, and pyruvate as carbon sources for proteins and nucleotide monophosphates for genes. Energy is derived from nucleoside monophosphates, which are regenerated in situ, rather than more expensive triphosphates. The instructions for protein or gene construction are contained in a plasmid, with a polymerase added for propagating the gene.

The company is currently working at the 100 L scale but there is no theoretical or practical reason why it cannot be scaled to 1,000 L or more. This volume, says Dr. Gold, could service demand for almost any therapeutic protein since the process can be repeated several times per week. A 1,000 L reaction would require extract harvested from a 2,000 L E. coli fermentation. The only limitation, which might eventually be overcome by coupling cell-free synthesis with other biotransformations, is that E. coli lack the machinery to glycosylate proteins.

What is remarkable is that Sutro achieves nearly seamless scalability without changing cell lines or reformatting, and with practically no process development in the traditional sense as one would expect with growing cells and microorganisms.

The process advantages for cell-free systems in large-scale protein production are potentially huge. Unit operations used in cell-free synthesis are scalable and familiar to those who work on microbial fermentation—mostly filtration and centrifugation. A single extract is suitable for production of any protein. And, unlike living cells, extracts are available for use on short notice. Since the E. coli extracts are “instructed” to produce only one protein, host cell protein issues generally disappear. Purer product streams mean leaner downstream separations as well.

“We believe that transforming biological reactions into a controllable enzymatic synthesis will revolutionize the way people look at protein manufacturing,” says Dr. Gold.

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